This program will assemble for the first time an HPLC and a continuous flow isotope ratio mass spectrometer. The component pieces are: 1. a high performance liquid chromatograph (HPLC); 2. a Vestec Universal HPLC/MS interface; 3. a chemical reaction interface (CRI) and 4. an isotope ratio mass spectrometer system (IRMS). This proposal emerges from several prior accomplishments. The first of these is the PI's development of the CRI, a microwave-induced reaction chamber that is placed between a chromatographic column and a mass spectrometer (CRI-MS). This apparatus has been used for many CRI-MS applications with capillary gas chromatography coupled to conventional mass spectrometers. More recently, a collaboration with Vestec has produced an interface to the CRI for HPLC that makes the approach outlined in the proposal possible. The orders of magnitude improvement provided by an IRMS in measuring low isotopic enrichments should expand the capabilities of CRI-MS. Furthermore, the unique chemistry of the CRI should provide 15N determinations that are superior to classical combustion methods. Compared to HPLC/conventional MS approaches, 13C and 15N will be selectively detected at greatly reduced isotopic abundance. An HPLC/IRMS offers researchers who use isotopes a significant expansion of target molecule types. After the assembly and technical evaluation of the HPLC/CRI-IRMS instrument, the bulk of the project will investigate just how much this range of applications has been expanded by evaluating a wide variety of compound classes. It is proposed that intact biological macromolecules can be analyzed for isotopic content directly by the CRI- IRMS. This will greatly improve analyses in biological systems where either 14C would have been a tracer or where the tedious sequence of hydrolysis followed by chromatographic separation and MS analysis of selected monomers would be required.